Glycosylated asterisks are among the most potent low valency inducers of Concanavalin A aggregation

Article abstract of DOI:10.1039/b814816c

A new class of sulfurated, semi-rigid, radial and low-valent glycosylated asterisk ligands with potential dual function as ligand and probe has some of the highest inhibition potencies of Con A-induced hemagglutination, by using a cross-linking mechanism of Con A which amplifies the enhancement to near nanomolar concentrations with the α-D-mannose asterisk. The Royal Society of Chemistry 2008.

Full text of DOI:10.1039/b814816c

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Glycosylated asterisks are among the most potent low valency inducers
of Concanavalin A aggregationw
b
a
Mazen Sleiman,z Annabelle Varrot,*c Jean-Manuel Raimundo,z Marc Gingrasz*a
and Peter G. Goekjian*b
Received (in Cambridge, UK) 26th August 2008, Accepted 30th September 2008
First published as an Advance Article on the web 7th November 2008
DOI: 10.1039/b814816c
A new class of sulfurated, semi-rigid, radial and low-valent
glycosylated asterisk ligands with potential dual function as
ligand and probe has some of the highest inhibition potencies
of Con A-induced hemagglutination, by using a cross-linking
mechanism of Con A which amplifies the enhancement to near
nanomolar concentrations with the a-D-mannose asterisk.
an alternating up and down pattern of the thiophenyl groups
above and below the plane of the central benzene ring,8 which
potentially could be used to pre-orient epitopes into particular
patterns.
In practice, we synthesized and evaluated a new class of
semi-rigid ligands based on a hexa-amino persulfurated ben-
zene asterisk 1,9 decorated with a-glucose, b-glucose and
a-mannose epitopes. The hexa-amino asterisk 1 was prepared
on a multi-gram scale, by coupling of sodium 4-acetamido-
thiophenolate with C6Cl6 in DMI and deprotection in
concentrated hydrochloric acid (93% overall yield; 412 g).
Coupling of the asterisk 1 to the glycoconjugate acids 2,10 3,11
and 412 of a-glucose, b-glucose and a-mannose, respectively,
was achieved using 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-
quinoline (EEDQ) as a coupling agent in yields ranging from
50 to 55% (Scheme 1). The deprotection of the glycosylated
Protein–carbohydrate interactions mediate a wide range of
intercellular communication processes, including cellular
adhesion.1 Although those interactions are often generated
by individual weak association constants of limited specifi-
city,2 the assembly of multiple carbohydrate–protein com-
plexes offers, through polyvalency, the avidity and selectivity
necessary for their biological function.3
The structure of the molecular scaffold, the mechanism and
effectiveness of the inhibition remains a central issue for
understanding multivalent interactions. Many glycoclusters,4
glycodendrimers5 and polyglycomers6 have been prepared in
order to delineate the structure/activity relationships of the
scaffold.3,6a,7
´
asterisks 5–7 proceeded smoothly under Zemplen conditions
to afford compounds 8–10 (Scheme 1).
A biochemical evaluation of the glyco-asterisks 8–10 was
investigated with Concanavalin A (Canavalia ensiformis, Con
A).13 This lectin interacts with cell surfaces by a process
implicating multivalent complexes with non-reducing
a-glucose and a-mannose residues. The inhibition of hemagglu-
tination with sugar-based ligands in the presence of Con A
has been widely used as a reference in carbohydrate–protein
interaction studies, and was thus chosen for this first evalua-
tion. In order to assess the activity of the glyco-asterisks, we
compared their ability to inhibit agglutination of rabbit
erythrocytes by Con A with that of their monovalent analo-
gues, Me-a-Glc, Me-a-Man, and the aniline amides, N-phenyl-
2-(a-D-Glc)acetamide 11 and N-phenyl-2-(a-D-Man)acetamide
Our contribution here relies on the concept of a spherical-
shaped scaffold with a dual function as a ligand and as a
probe, combined with the need for semi-rigidity and amplifi-
cation of inhibition through Con A/ligand auto-assemblies
under kinetic conditions. The asterisks were selected as
scaffolds/probes based on their synthetic, physical, and con-
formational properties. They are well-defined and have
demonstrated potentially useful properties (redox potentials,
UV/Vis absorption, conductivity, etc.)8 which could facilitate
detection by colorimetric, electrochemical and spectroscopic
methods. They usually exhibit a conformational preference for
a Interdisciplinary Center on Nanoscience of Marseille (CINaM),
´
´
´
UPR CNRS 3118, (affiliated with Universite de la Mediterranee),
Campus de Luminy, Case 913, Marseille, Cedex 9, France.
E-mail: marc.gingras@univmed.fr; Fax: (+33)491829155;
Tel: (+33)491829155
b Laboratoire de Chimie Organique II-Glycochimie, UMR CNRS
´
ˆ
5246, Universite Claude Bernard – Lyon 1, C.P.E. Lyon, Batiment
308D, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne
Cedex, France. E-mail: goekjian@univ-lyon1.fr;
Fax: (+33) 478898914; Tel: (+33) 472448183
´
c
´ ´
Centre de Recherches sur les Macromolecules Vegetales
´
(CERMAV), UPR CNRS 5301 (affiliated with Universite Joseph
Fourier), 601 rue de la Chimie, BP 53, 38041 Grenoble Cedex 9,
France. E-mail: annabelle.varrot@cermav.cnrs.fr
w Electronic supplementary information (ESI) available: Experimental
section. See DOI: 10.1039/b814816c
z Former address: Institut de Chimie de Nice, Faculte
Universite de Nice-Sophia Antipolis, 28 avenue Valrose, 06108 Nice
Cedex 2, France.
´
des Sciences,
´
Scheme 1
ꢀc
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Chem. Commun., 2008, 6507–6509 | 6507

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Table 1 Hemagglutination inhibition assays for 8–10 with Con A
Ligand
IC50a/mM
bb
b/Nc
Me-a-D-glucopyranoside
N-Phenyl-2-(a-D-glucopyranosyloxy)acetamide 11
8 (a-Glc)
4.0
9.9
0.011
NI at 11.7
2.0
1
1
0.4
364
—
0.4
61
—
1
0.8
3750
9 (b-Glc)
Me-a-D-mannopyranoside
N-Phenyl-2-(a-D-mannopyranosyloxy)acetamide 12
10 (a-Man)
1
2.5
0.8
22 472
8.9 ꢁ 10ꢂ5
a
b
c
Each value represents the average of at least three assays. Enhancement values b = IC50(monomer)/IC50(cluster). Relative enhancement per
sugar unit b/N.
12, and as a negative control, the hexavalent b-glucose asterisk
9, whose sugar moiety is not recognized by the lectin (Table 1).
After careful blank control experiments and several repro-
duceable assays, the a-glucose and a-mannose asterisks 8 and
10 showed a strongly enhanced inhibition in both cases relative
to their monovalent analogues. The a-glucose asterisk 8
inhibited hemagglutination at 11 mM, which corresponds to
a 60-fold enhancement per sugar relative to Me-a-glucoside.
On the other hand, the a-mannose asterisk 10 showed one of
the best reported inhibitory potency at a minimum concentra-
tion of 89 nM, and hence greater than 3750-fold increase in
relative activity per sugar compared to Me-a-mannoside. Two
control experiments indicated that non-specific interactions of
the scaffold were not responsible for the inhibition effect: the
low enhancement values (b) for the aniline amides exclude sub-
site assisted binding of the sugar by the amide or aromatic
group;14 the b-glucose asterisk 9, the most structurally analo-
gous compound available, did not show any inhibition even at
11.7 mM concentration. The high enhancement of inhibition
by 8 and 10, in comparison to their monovalent analogues,
indicates a powerful cross-linking ability.
Fig. 1 DLS experiments on a-Man asterisk 10 with Con A. (a) Red:
Con A 17 mM in 450 ml of HEPES 20 mM, pH 7.2, 150 mM NaCl,
2 mM CaCl2, 2 mM MnCl2: solution A. (b) Green: A + 50 nM of 10;
(c) Blue: A + 100 nM of 10. (d) Black: A + 150 nM of 10. (e) Purple:
A + 200 nM of 10. (f) Pink: A + 400 nM of 10.
exclusively an aggregated species with a hydrodynamic radius
near 850 nm (blue curve). This process is mannose-dependent
and is partially reversible. A high concentration of Me-a-Man
is required to offset the effect of the asterisk. Adding increasing
concentrations of Me-a-Man (0, 1, 10, 500, 1000 mM) to a
series of solutions containing Con A (17 mM) and 10 (0.5 mM)
showed reversion of the aggregation only above 1 mM of
Me-a-Man. It is noteworthy that identical solutions mixed in a
different order did not give the same final result, even after
several hours. These results suggest that, rather than an
increased avidity for Con A, the unusual potency of the
asterisks is due to an exceptionally efficient, kinetically con-
trolled aggregation process that strongly amplifies the effect of
these low valency ligands.
In addition, there is a 60-fold amplification of the selectivity
between the mannose and glucose asterisks relative to the
monovalent compounds. While in the monovalent case,
Me-a-Man inhibits at only a two-fold lower concentration
than Me-a-D-glucoside, the mannose asterisk 10 inhibits at a
120-fold lower concentration than its glucose analogue 8.
A 100-fold selectivity has rarely been reported, except in the
case of polyvalent Man/Glc polyglycomer scaffolds.15
In conclusion, we have successfully validated several new
concepts with the asterisk scaffolds and Con A. We synthe-
sized a new class of circular, semi-rigid glycosylated sulfurated
asterisks which have some of the highest inhibition potencies
of Con A-induced hemagglutination, in addition to their
potentially useful optical and electrophysical properties.
A complete, rapid, and sugar-dependent aggregation of
Con A at such low concentrations (89 nM) has rarely been
reported with low-valency clusters.18 DLS data indicated a
cross-linking mechanism capable of aggregating Con A near
the nanomolar range for the a-mannose asterisk. This activity
can be attributed to a powerful cross-linking effect of the
asterisk, which sequesters the lectin in a macromolecular
assembly,7 resulting in an amplification of the inhibition. It
seems unusually favorable, compared to other systems of
similar valency and our current hypothesis is based on the
ability of semi-rigid structures to optimally adjust and prop-
erly present the sugar epitopes to the receptor for the rapid
formation of a crosslinked macromolecular complex.17 In an
encouraging result, we have observed similar potencies with
In a nutshell, the a-O-mannosylated asterisk 10 has one
of the highest reported enhancements (b = 22 472) and
selectities (IC50 8/IC50 10 = 120) to date for inhibition of
hemagglutination induced by Con A, amongst discrete low
valency ligands.
A visual turbidity test showed a rapid precipitation of Con
A in the presence of an equimolar amount of the asterisk.16
This result suggests that the semi-rigid architecture of the
asterisk plays an important role in inducing a strong cross-
linking of Con A.6a,17 Dynamic light scattering (DLS) experi-
ments16 confirmed a strong aggregating effect on Con A by 10
at concentrations slightly above the nanomolar range. Fig. 1
shows a series of solutions containing Con A (17 mM) and 10
at different concentrations (0, 50, 100, 150, 200 and 400 nM).
Without 10 a distribution centered around a hydrodynamic
radius of 3 nm (red curve) was observed. A 100 nM concen-
tration of the asterisk 10 induced a dramatic shift to nearly
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more biologically relevant lectins, using similar asterisks, and
the results will be reported in due course.
2002, 90, 291–309D. Zanini and R. Roy in Carbohydrate Mimics:
Concepts and Methods, ed. Y. Chapleur, Verlag Chemie,
Weinheim, Germany, 1998, pp. 385–415.
M. S. thanks The Islamic Center of Higher Education
(Lebanon), the UCBL-1, and the University of Nice for a
PhD scholarship. The CNRS and French Ministry of Research
(MENESR) are thanked for financial support. We gratefully
acknowledge the animal center of IUTA-Lyon, as well as
Dr. Hans J. Raeder and Ali Rouhanipour of the Max Planck
Institute (Mainz, DE) and Jean-Marie Guigonis, IFR 50,
Faculty of Medicine Pasteur (Nice) for mass spectral analyses.
8 M. Gingras, J. M. Raimundo and Y. M. Chabre, Angew. Chem.,
Int. Ed., 2006, 45, 1686–1712.
9 For other asterisk structures: J. Kim, Y. Ahn, K. M. Park, Y. Kim,
Y. H. Ko, D. H. Oh and K. Kim, Angew. Chem., Int. Ed., 2007, 46,
7393–7395; A. Dondoni, A. Marra and M. G. Zampolli, Synlett,
2002, 1850–1854; R. Dominique, B. Liu, S. Das and R. Roy,
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Schuster and L. L. Kiessling, J. Am. Chem. Soc., 2000, 122,
4518–4519.
10 Acid glycoconjugates 2–4 were prepared in good yields from the
corresponding allyl glycosides by KMnO4 oxidation of the double
bond.
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